Boyd E. Haley

Chemical Attachment of Organic Functional Groups to Single-walled Carbon Nanotube Material

We have subjected single-walled carbon nanotube materials (SWNTMs) to a variety of organic functionalization reactions. These reactions include radioactive photolabeling studies using diradical and nitrene sources, and treatment with dichlorocarbene and Birch reduction conditions. All of the reactions provide evidence for chemical attachment to the SWNTMs, but because of the impure nature of the staring materials, we are unable to ascertain the site of reaction. In the case of dichlorocarbene we are able to show the presence of chlorine in the SWNT bundles, but as a result of the large amount of amorphous carbon that is attached to the tube walls, we cannot distinguish between attachment of dichlorocarbene to the walls of the SWNTs and reaction with the amorphous carbon.

Abnormal properties of creatine kinase in Alzheimer's disease brain : Correlation of reduced enzyme activity and active site photolabeling with aberrant cytosol-membrane partitioning

The report shows that Alzheimers disease (AD) brain creatine kinase (CK) is modified such that the nucleotide binding site of CK is blocked and that abnormal partitioning of CK between the soluble and pellet fractions occurs. First, CK activity was 86% decreased in AD brain homogenates in comparison to age-matched controls. Secondly, over a 23.5 fold greater 32P photoincorporation of [alpha 32P]8N3ATP was observed into CK of control vs. AD samples. Also, a 7.4-fold increase of enzyme induced 32P incorporation was observed in controls vs. AD samples by incubation with [gamma 32P]ATP. Thirdly, Western blot analysis showed that CK copy numbers in the AD homogenate were decreased by less than 14% in comparison to controls. However, analysis showed that control supernatant and pellet fractions contained 10.3 and 0.4 times the CK copy number found in the corresponding AD fractions. 32P incorporation by both photolabeling and enzyme catalyzed incorporation of radiolabel followed CK activity and not CK copy number. Further, [alpha 32P]ADP and [gamma 32P]ATP incorporated 32P into control brain and purified brain CK equally well, indicating that a mechanism different from gamma-phosphoryl transfer is involved in the enzymatic incorporation of radiolabel. Also, the level of abnormal partitioning of CK into AD brain pellet correlated with the decreased [32P]8N3GTP photolabeling and abnormal partitioning of beta-tubulin, a protein known to be aberrantly modified in the AD brain. This indicates that a common chemistry is affecting both CK and tubulin in AD.

[58] Synthesis and use of azido photoaffinity analogs of adenine and guanine nucleotides

Publisher Summary This chapter discusses the synthesis and use of azido photoaffinity analogs of adenine and guanine nucleotides. Numerous reagents have been used to chemically modify enzymes and identify amino acid residues that are necessary for enzyme catalysis. However, most of these reagents are nonspecific compounds that react with any available functional group on the enzyme with little or no selectivity for groups located within the active site. To label the active site with some degree of selectivity, a specific affinity reagent should be used. These affinity reagents should contain most of the structural properties of the natural occurring substrate and an added reactive group, which has the potential to form an irreversible covalent bond with an amino acid residue located within the active site. Many potential active site-directed affinity analogs of ATP and GTP have been synthesized. A little success has been achieved in the area of “specific covalent labeling” of ATP and GTP binding sites, because the binding site must contain an amino acid residue that has the potential to form a covalent bond with a reactive group on the nucleotide analog.

Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.

Thimerosal Exposure and the Role of Sulfation Chemistry and Thiol Availability in Autism

Autism spectrum disorder (ASD) is a neurological disorder in which a significant number of the children experience a developmental regression characterized by a loss of previously acquired skills and abilities. Typically reported are losses of verbal, nonverbal, and social abilities. Several recent studies suggest that children diagnosed with an ASD have abnormal sulfation chemistry, limited thiol availability, and decreased glutathione (GSH) reserve capacity, resulting in a compromised oxidation/reduction (redox) and detoxification capacity. Research indicates that the availability of thiols, particularly GSH, can influence the effects of thimerosal (TM) and other mercury (Hg) compounds. TM is an organomercurial compound (49.55% Hg by weight) that has been, and continues to be, used as a preservative in many childhood vaccines, particularly in developing countries. Thiol-modulating mechanisms affecting the cytotoxicity of TM have been identified. Importantly, the emergence of ASD symptoms post-6 months of age temporally follows the administration of many childhood vaccines. The purpose of the present critical review is provide mechanistic insight regarding how limited thiol availability, abnormal sulfation chemistry, and decreased GSH reserve capacity in children with an ASD could make them more susceptible to the toxic effects of TM routinely administered as part of mandated childhood immunization schedules.

The relationship between mercury and autism: A comprehensive review and discussion.

The brain pathology in autism spectrum disorders (ASD) indicates marked and ongoing inflammatory reactivity with concomitant neuronal damage. These findings are suggestive of neuronal insult as a result of external factors, rather than some type of developmental mishap. Various xenobiotics have been suggested as possible causes of this pathology. In a recent review, the top ten environmental compounds suspected of causing autism and learning disabilities were listed and they included: lead, methyl-mercury, polychorinated biphenyls, organophosphate pesticides, organochlorine pesticides, endocrine disruptors, automotive exhaust, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, and perfluorinated compounds. This current review, however, will focus specifically on mercury exposure and ASD by conducting a comprehensive literature search of original studies in humans that examine the potential relationship between mercury and ASD, categorizing, summarizing, and discussing the published research that addresses this topic. This review found 91 studies that examine the potential relationship between mercury and ASD from 1999 to February 2016. Of these studies, the vast majority (74%) suggest that mercury is a risk factor for ASD, revealing both direct and indirect effects. The preponderance of the evidence indicates that mercury exposure is causal and/or contributory in ASD.

Methodological Issues and Evidence of Malfeasance in Research Purporting to Show Thimerosal in Vaccines Is Safe

There are over 165 studies that have focused on Thimerosal, an organic-mercury (Hg) based compound, used as a preservative in many childhood vaccines, and found it to be harmful. Of these, 16 were conducted to specifically examine the effects of Thimerosal on human infants or children with reported outcomes of death; acrodynia; poisoning; allergic reaction; malformations; auto-immune reaction; Wells syndrome; developmental delay; and neurodevelopmental disorders, including tics, speech delay, language delay, attention deficit disorder, and autism. In contrast, the United States Centers for Disease Control and Prevention states that Thimerosal is safe and there is “no relationship between [T]himerosal[-]containing vaccines and autism rates in children.” This is puzzling because, in a study conducted directly by CDC epidemiologists, a 7.6-fold increased risk of autism from exposure to Thimerosal during infancy was found. The CDCs current stance that Thimerosal is safe and that there is no relationship between Thimerosal and autism is based on six specific published epidemiological studies coauthored and sponsored by the CDC. The purpose of this review is to examine these six publications and analyze possible reasons why their published outcomes are so different from the results of investigations by multiple independent research groups over the past 75+ years.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Nucleotide photoaffinity labeling of protein kinase subunits

Publisher Summary This chapter presents a series of experimental considerations that may be used to resolve problems faced when using nucleotide photoaffinity probes to detect the subunits involved in the catalytic activity of protein kinases. The ideas presented are general in nature, since each different nucleotide-binding protein have its own characteristics that must be considered when designing the initial photolabeling experiments. It is important to realize that photoaffinity labeling is very dependent on binding affinity and that proteins with K d values in the millimolar range are less likely to be good candidates for photolabeling than those with micromolar K d values. Also, it is important to consider the turnover number of the nucleotide-hydrolyzing enzymes since it is obviously difficult to photolabel an enzyme that is hydrolyzing the probe faster than one can finish the photolysis. Likewise, enzymes that autophosphorylate rapidly can be difficult to photolabel with γ- 32 P-labeled probes under conditions where the enzyme is active. With such enzymes the use of the γ- 32 P-labeled probe is preferred unless autophosphorylation can be slowed to a minimum by decreasing the temperature or changing metal ions. Consideration for the metal ion requirements is a must. Usually, if the catalytic activity requires Mg 2+ , then the binding and subsequent photoinsertion would also require the presence of a divalent metal ion. If the entire goal of the experiment is to determine which protein of a complex mixture is binding a particular nucleotide this may be accomplished by addition of the photoprobe to concentrations that saturate the binding site as approximated by the predetermined K d of the enzyme for its native substrate.

New science challenges old notion that mercury dental amalgam is safe

Mercury dental amalgam has a long history of ostensibly safe use despite its continuous release of mercury vapor. Two key studies known as the Children’s Amalgam Trials are widely cited as evidence of safety. However, four recent reanalyses of one of these trials now suggest harm, particularly to boys with common genetic variants. These and other studies suggest that susceptibility to mercury toxicity differs among individuals based on multiple genes, not all of which have been identified. These studies further suggest that the levels of exposure to mercury vapor from dental amalgams may be unsafe for certain subpopulations. Moreover, a simple comparison of typical exposures versus regulatory safety standards suggests that many people receive unsafe exposures. Chronic mercury toxicity is especially insidious because symptoms are variable and nonspecific, diagnostic tests are often misunderstood, and treatments are speculative at best. Throughout the world, efforts are underway to phase down or eliminate the use of mercury dental amalgam.